Commensal bacteria are usually regarded as beneficial to the host by displacing pathogens from a microbial niche or providing protection by continually stimulating epithelial surfaces to express and secret antimicrobial peptides (AMPs) at levels that kill opportunistic/pathogenic organisms (4
). The release of antimicrobial chemokine CCL20 (51
) requires stimulation for its release, and the discrete stimuli that lead to its upregulation may be cell specific (17
). In these studies, we demonstrated the ability of primary oral epithelial cells to release CCL20 in response to the oral commensal bacterium F. nucleatum
and other epithelium-derived agents that it also induces, i.e., hBD-2, hBD-3, TNF-α, and IL-1β. The kinetic studies of CCL20 release by HOECs suggested that this is an early event subsequent to challenge by any of the agents used. FnCW caused rapid release of CCL20 from HOECs, i.e., 6 h postchallenge there was a 6-fold-greater presence of CCL20 in the spent supernatant than control supernatant and twice the increase of a 24-h challenge (6-fold versus ~3-fold). This is in agreement with a recent study that showed that F. nucleatum
elicits rapid induction of the CCL20 gene (72.7-fold in 6 h compared to 21.7-fold in 24 h) (62
). Lin et al. (43
) also showed early release of CCL20 by human mast cells in response to Pseudomonas aeruginosa
, which also peaked at 6 h. Adenylate and uridylate (A+U)-rich element (ARE)-mediated mRNA turnover is an important regulatory component of gene expression for innate and specific immunity (24
). The transient and rapid upregulation of CCL20 gene expression by FnCW could be due to posttranscriptional regulation of mRNA stability via AREs (19
). Indeed, when we looked for AREs within the 3′-untranslated region of CCL20 mRNA, using the ARE website (http://rna.tbi.univie.ac.at/AREsite
), we found three ATTTA domains (see Fig. S1 in the supplemental material). Another rapidly induced gene by FnCW in HOECs is CXCL5 (40.5-fold in 6 h) (62
), and by using the ARE site we found 11 ATTTA domains within its 3′-UTR (see Fig. S1). We could not find any AREs within the 3′-UTR of DEFB4 (hBD-2) or in DEFB103 (hBD-3) and, not surprisingly, these defensins were not released as rapidly as CCL20 by FnCW in HOECs.
In the present study, we found that actinomycin D and cycloheximide inhibited FnCW-induced release of CCL20 by HOECs, indicating that CCL20 release requires transcriptional and translational activation. BFA is a fungal metabolite able to rapidly and reversibly block intracellular vesicle transport from the endoplasmic reticulum to the Golgi apparatus and, therefore, inhibit protein secretion with minimal effect on protein synthesis (38
). BFA blocks the release of newly synthesized proteins but has no effect on preformed granule-bound proteins (45
). Although BFA has been widely used as an inhibitor of protein secretion, the information concerning its effect on CCL20 release has not been reported. Our study showed that BFA inhibited not only the FnCW-induced release of CCL20 by HOECs but also constitutive (unstimulated) release of CCL20. The increased levels of intracellular CCL20 in unstimulated and FnCW-stimulated cell lysates from BFA-pretreated HOECs further corroborate the inhibition of CCL20 release by BFA. Given the well-characterized properties of BFA, it is reasonable to conclude that CCL20 is secreted through the vesicle transport pathway.
Certain commensal bacteria are excellent inducers of beta-defensins (22
), suggesting that the commensal bacterial community may act by priming innate immune readiness of the oral epithelium. We and others have shown that F. nucleatum
can induce innate response elements, such as hBDs, in HOECs (29
). More recently, several immunoregulatory functions within the adaptive immune system have been attributed to hBDs, in addition to their antimicrobial capacity, i.e., their ability to cross talk with the adaptive immune system by acting as chemokines (34
) and by changing a cell's phenotype, either with activation, as seen with antigen-presenting cells (25
), or antagonism, as seen with T cells (21
). These findings conform to reports in the literature that innate responses usually precede, and are necessary for, the establishment of adaptive immunity (44
). Upon stimulation by antimicrobial peptides, different immune and inflammatory cells have been shown to produce cytokines and/or chemokines. For example, the alpha-defensins human neutrophil peptide 1 and 3 stimulate the production of IL-1, -4, and -6, TNF-α, and gamma interferon in monocytes (5
). Moreover, hBDs and LL-37 have been reported to enhance the generation of IL-18 in keratinocytes (47
). In this study, we demonstrated that the inducible defensins hBD-2 and hBD-3 can induce the release of other AMPs/chemokines, i.e., CCL20, by HOECs. Therefore, the ability of hBDs to promote the release of the antimicrobial chemokine CCL20 suggests the broad role of human antimicrobial peptides in primary immune responses by oral epithelial cells. This regulatory loop could act in a complementary manner to provide added protection against microbial assault.
To gain initial insight into the cellular pathways through which defensins induce CCL20 release by HOECs, we focused on three major downstream MAPK cascades: ERK, p38, and JNK. In cells, these kinase cascades act as signal sorters for a variety of upstream signals before entering the nucleus. The p38, ERK, and JNK kinase cascades have been shown to be involved in a large variety of cellular activities (3
). Defensins have been shown to activate primary human keratinocytes through p38 and ERK1/2 (47
). By focusing on these three MAPKs, we demonstrated that both ERK1/2 and p38, but not JNK, are required for hBD-induced CCL20 secretion by HOECs. The involvement of similar MAPKs was also observed in the release of CCL20 by TNF-α- or IL-1β-stimulated oral epithelial cells. The p38 MAPK pathway plays an important role in the posttranscriptional regulation of inflammatory genes and has been found to regulate both the translation and the stability of inflammatory mRNAs. The mRNAs regulated by p38 are known to have AREs present in their 3′-untranslated regions (16
) and thus, the presence of an ARE within the 3′-UTR of CCL20 supports the involvement of p38 in its induction by defensins and cytokines. Surprisingly, ERK1/2 and p38 inhibitors were found to have a negligible effect on FnCW-induced release of CCL20 by HOECs. Most recently, Yin and Chung (63
) found a lack of involvement of p38 in F. nucleatum
-induced CCL20 mRNA induction. At this stage, however, it is not clear why, in contrast to induction of CCL20 by defensins and cytokines, the induction of CCL20 by FnCW does not occur via p38, despite the fact that FnCW is capable of causing rapid and transient activation of p38 MAPK (42
) in HOECs. Nevertheless, these observations further corroborate the fact that the induction of CCL20 by FnCW is not dependent on defensins (hBD-2 and -3), TNF-α, or IL-1β.
Kinetics of FnCW-induced CCL20 and β-defensin release are distinct, i.e., CCL20 release peaks at 4 to 6 h () by FnCW, while the peak time is 24 h for hBD-2 () and 48 h for hBD-3 (). Thus, coordinated stimulation of CCL20 and beta-defensins by FnCW on oral epithelial cells might suggest a complementary activity in exerting the innate nature of F. nucleatum
in host defense. Since FnCW is a complex mixture of proteins, it is tempting to speculate that different components within the FnCW fraction are responsible for the release of beta-defensins and CCL20. We have recently identified a Fusobacterium
-associated beta-defensin inducer peptide (FAD-I) within FnCW that is responsible for the induction of hBD-2 in HOECs (29
). When we tested FAD-I's ability to induce CCL20 release by HOECs, we found it to be negligible compared to FnCW (), while its ability to induce hBD-2 release was comparable to that of FnCW (). However, an isoelecric focusing (IEF) fraction of crude FnCW, from which FAD-I was further isolated (29
), was capable of inducing CCL20 release by HOECs (), indicating that the CCL20 inducing factor(s) is present in the IEF fraction. The results shown in and B appear to suggest this. Although the IEF fraction was a semipurified fraction from crude FnCW, the phosphorylation patterns of different MAPKs elicited by the IEF fraction were similar to that elicited by crude FnCW () and, not surprisingly, the effectiveness of the IEF fraction in inducing CCL20 or hBD-2 was close to that of FnCW. Isolation of the CCL20-inducing factor, in conjunction with FAD-I, may one day be used to bolster the innate defenses of vulnerable mucosae.
Fig. 9. Comparison of FnCW, IEF fraction of FnCW-induced and recombinant FAD-I (rFAD-I)-induced CCL20 and hBD-2 release. HOECs (mixed donors; N = 3) were treated with medium alone, FnCW (10 μg/ml), IEF fraction (10 μg/ml), or rFAD-I (10 μg/ml) (more ...)
Fig. 10. PathScan MAPK multitarget sandwich ELISA results. HOEC monolayers (mixed donors; N = 3) were treated with either IEF (10 μg/ml) or FnCW (10 μg/ml) for 0, 30, and 60 min. The cell lysates were analyzed for the indicated target, using the (more ...)